Multicellularity enables the growth of complex life forms as it allows for specialization of cell types, differentiation, and large scale spatial organization. In a similar way, modular construction of synthetic multicellular systems will lead to dynamic biomimetic materials that can respond to their environment in complex ways. In order to achieve this goal, artificial cellular communication and developmental programs still have to be established. Here, we create geometrically controlled spatial arrangements of emulsion-based artificial cellular compartments containing synthetic in vitro gene circuitry, separated by lipid bilayer membranes. We quantitatively determine the membrane pore-dependent response of the circuits to artificial morphogen gradients, which are established via diffusion from dedicated organizer cells. Utilizing different types of feed-forward and feedback in vitro gene circuits, we then implement artificial signaling and differentiation processes, demonstrating the potential for the realization of complex spatiotemporal dynamics in artificial multicellular systems.

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.

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A molecular engineering strategy based on rational variations of the bromine substitution pattern in two-photon absorbing singlet oxygen sensitizers allows studying the relations that exist between the positioning of an inter-system crossing promoter on the charge-transfer chromophore and its ability to generate singlet oxygen.

Compartmentalizationa nd spatial organization of biochemical reactionsa re essential for the establishment of complex metabolic pathways inside synthetic cells. Phospholipid and fatty acid membranes are the most natural candidates for this purpose, but also polymers have shown great potential as enclosureso fa rtificial cell mimics.H erein, we reporto nt he formation of giant vesiclesi nasize range of 1 mm-100 mmu sing amphiphilic elastin-like polypeptides. The peptide vesicles can accommodatec ell-freeg ene expression reactions, which is demonstrated by the transcription of af luorescent RNA aptamer and the production of af luorescent protein. Importantly,g ene expression inside the vesicles leads to a strong growth of their size-upto an order of magnitude in volumei ns everal cases-which is driven by changes in osmotic pressure, resulting in fusion eventsa nd uptake of membrane peptides from the environment.

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.

Biomaterials composed of synthetic cells have the potential to adapt and differentiate guided by physicochemical environmental cues. Inspired by biological systems in development, which extract positional information (PI) from morphogen gradients in the presence of uncertainties, we here investigate how well synthetic cells can determine their position within a multicellular structure. To calculate PI, we created and analyzed a large number of synthetic cellular assemblies composed of emulsion droplets connected via lipid bilayer membranes. These droplets contained cell-free feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. PI is found to be limited by gene expression noise and affected by the temporal evolution of the morphogen gradient and the cell-free expression system itself. The generation of PI can be rationalized by computational modeling of the system. We scale our approach using three-dimensional printing and demonstrate morphogen-based differentiation in larger tissue-like assemblies.

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